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Sample GSM697064 Query DataSets for GSM697064
Status Public on Mar 23, 2011
Title Cl8 CNV extraction1_seq1 aliquote 1
Sample type SRA
 
Source name Cl8 CNV extraction1_seq1 channel_1
Organism Drosophila melanogaster
Characteristics cell line: CME-W1-Cl.8+
tissue: dorsal mesothoracic disc
developmental stage: third instar larval stage
Sex: Male
Growth protocol Cell growth protocol; Grow 10ml of cells in 100mm dishes For Kc and S2 cells use : Schneider's medium (invitrogen 11720-067) 10% ModEncode serum 1% Penicillin/Streptomycin/Glutamine (PSG) (Invitrogen 10378-016) small edit For ML-DmBG3-c2 cells use: Schneider's medium 10% ModEncode serum 1% PSG 10ug/ml human insulin (sigma I9278)) Cells are grown at 25C. Cells are happiest between 1.5X106 and 2.0X106
Extracted molecule genomic DNA
Extraction protocol DNA isolation protocol; 1.Grow 10ml of cells to 1.5 x 106 in 100mm dishes 2.Harvest cells into 15ml conical tubes. 3.Pellet cells at 1000g for 5 minutes 4.Wash cells once in cold 1X PBS 5.Resuspend pellet in 0.7ml 10mM Tris pH 9.5 6.Add 0.7ml NDS. Invert to mix 7.Add 0.1ml 20mg/ml Proteinase K (Amresco 0706-1G). Invert to mix 8.Inc. 37C, 2 hours. 9.Add 1.5ml 1X TE. Invert to mix 10.Add 3.0ml phenol/chloroform. Invert tubes over 5 minutes 11.Centrifuge 3500 rpm for 10-12 minutes 12.Remove top layer with cut p1000 tips to a new 15ml tube 13.Repeat steps 10-12. 14.Add 2.5 volumes of room temp. 100% Ethanol. Invert 15.Inc. at room temp overnight 16.Centrifuge tubes 3500-3700rpm for 10-12 minutes 17.Wash pellet with 70% Ethanol and transfer pellet to a 1.5ml tube 18.Centrifuge full speed for 5 minutes 19.Air dry pellet 20.Resusp. pellet in 250ul 1X TE 21.Add 5ul 10mg/ml RNaseA (Amresco 0675-250mg). Inc. 37C for 1h 22.Add 1/10 volume 3M NaOAc. Vortex 23.Add 2X volume cold 100% Ethanol. Vortex 24.Inc. -80C 20-30 minutes 25.Centrifuge at 4C for 15 minutes 26.Wash with 70% Ethanol 27.Air dry pellet. Resusp. in 250ul 1X TE 28.Spec DNA Shearing DNA 29.Shear DNA 3X 10 seconds with 1minute on ice in between.
Genomic DNA library protocol; Genomic DNA Library 1. Genomic DNA was fragmented to less than 800bps by sonication 2. the library was prepared as described in the Illumina provided protocol "Preparing samples for sequencing genomic DNA" (part # 11251892 Rev. A) with the exception of an additional gel extraction (size select for 150-200bps) after the "enrich the adapter-modified DNA fragments by PCR" step. 3. Samples were submitted to the Duke University IGSP sequencing facility.
 
Library strategy WGS
Library source genomic
Library selection RANDOM
Instrument model Illumina Genome Analyzer
 
Description In this experiment, there is no ChIP step. Simply separate the polytene dna from the tissue (cell line) and seq them, if more reads (than other regions) then they are the CNV regions.
Data processing Illumina_sequencing:DM:1 protocol. Load sample onto flow cell at a (usually 2-8 pM, variable) concentration and run on an Illumina Genome Analyzer II for single-end sequencing for 36 or 76 nt. Image data is then deconvoluted using the most current versions of Firecrest, Bustard, and Gerald (or equivalents). Processed data are obtained using following parameters: read length is 36 Illumina_seq_maq_processing:DM:1 protocol. Illumina seq maq processing protocol; * Get Illumina FASTQ files back from Duke IGSP Sequencing Facility. Their steps, briefly: * Image analysis (SCS real time analysis software / Firecrest software) * Base calling ( Bustard.py) * Sequence analysis (GERALD.py) -> results in the FASTQ file that gets sent to us. * Using maq command-line tool v. 0.7.1: * Convert Illumina FASTQ file to Sanger-style FASTQ format: maq sol2sanger $workingdir/$solfastqfilename $workingdir/$solfastqfilename.sangerfastq * Convert Sanger FASTQ file to Binary FASTQ format: maq fastq2bfq $workingdir/$solfastqfilename.sangerfastq $workingdir/$solfastqfilename.sangerbfq * Convert Drosophila 5.1 FASTA file to Binary FASTA format: maq fasta2bfa $workingdir/$referencefastafile $workingdir/$referencefastafile.bfa * Map reads back to Drosophila genome allowing 3 mismatches: maq map -n 3 $workingdir/$destination_table.map $workingdir/$referencefastafile.bfa $workingdir/$solfastqfilename.sangerbfq * Create a mapview file to import into the database: maq mapview $workingdir/$destination_table.map > $workingdir/$destination_table.mapview * Processed data are obtained using following parameters: genome version is 5.1 CNV_seq_analysis:DM:1 protocol. CNV seq analysis protocol; * Read locations are shifted: * Positive reads are shifted downstream by half of the estimated sonication size. * Negative reads are shifted upstream by half of the estimated sonication size. * Reads with a map score (Phred) >= 35 (leading to maximum probability that a read is misaligned of ~0.03%) are then binned based on their location, with non-overlapping bins of size 1000bp covering the genome. * The total number of reads in each bin are summed across replicates.
 
Submission date Mar 23, 2011
Last update date May 15, 2019
Contact name DCC modENCODE
E-mail(s) help@modencode.org
Phone 416-673-8579
Organization name Ontario Institute for Cancer Research
Lab modENCODE DCC
Street address MaRS Centre, South Tower, 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
 
Platform ID GPL9058
Series (1)
GSE28136 CNV CME W1 Cl.8+ Cells
Relations
SRA SRX052711
BioSample SAMN00254002

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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